Raman spectroscopy is becoming an increasingly practical technique because of its minimal sample preparation requirements and compatibility with biological materials in aqueous solutions (E. E. Lawson, B. W. Barry, A. C. Williams, and H. G. M. Edwards, J. Raman Spectrosc. 28, 111 (1997); C. Krafft, Anal. Bioanal. Chem. 378, 60 (2004)). Surface-enhanced resonance Raman spectroscopy (SERRS) provides unprecedented enhancement, making it an attractive technique for applications in protein, nucleic acid, and related biomarker analysis (K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari, and M. S. Feld, Chem. Rev. 99, 2957 (1999)). SERRS not only overcomes the gap between the inherent sensitivity of Raman scattering and fluorescence, but the Raman spectral features are also larger (and sharper) than fluorescence from the same chromophore (A. Campion and P. Kambhampati, Chem. Soc. Rev. 27, 241 (1998)).
However, SERRS suffers from variability in enhancement of Raman intensity depending upon the nanomorphology of the substrate (typically silver colloids), thereby affecting the reproducibility of the measurement. Several approaches to addressing problems associated with the reproducibility and optimization of SERRS have been reported. One strategy involves the linkage of SERRS active molecules and analyte-binding molecules with the surface of SERRS enhancing particles (U.S. Patent Publication No. 2005/0089901). The correction of the variation of SERRS signals is also accomplished using either an internal or an external standard. The use of an isotopic-edited internal standards (IEIS) method was reported to improve the performance of SERRS with unprecedented accuracy (Zhang, D. M., et al. Anal. Chem. 2005, 77, 3563-69). The internal standardization prototype study used two isotopic variants of the rhodamine 6G (R6G) chromophore.
SERRS active molecules have been employed as labeling reagents for bioanalytical applications. Several classes of organic dyes have been reported as SERS active molecules since they exhibit high Raman cross-sections and offer single molecule detection. The most widely used dyes are rhodamine (xanthene class), crystal violet (triarylmethane class), and nile blue (oxazine class). Single molecule detection limits have been reported for rhodamine 6G, crystal violet, nile blue, and other SERRS active molecules (Kneipp, K., et al., Phys. Rev. E, 1998, 57, R6281-4; Koo, T.-W., et al., Appl. Spectrosc. 2004, 58, 1401-7; Nie, S., et al., Science, 1997, 275, 1102-6; Kneipp, K., et al., Phys. Rev. Lett. 1997, 78, 1667-70).
A number of studies have been undertaken on SERRS or SERS for biomolecular detection. For example, Mirkin et al. (U.S. Patent Publication No. 2003/0211488) employed a dye-conjugated gold particle for identification of target DNA sequences. Rhodamine 6G-NHS ester derivative was used to label oligonucleotides. Also a bar coding strategy using multiple dyes created unique labels. However, isotopic variants of R6G were not disclosed in this patent, nor was the composition of dye envisioned for determination of analyte quantities.
Chaiken, J. et al. (U.S. Pat. No. 6,503,478) disclosed deuterated labeling agents and their Raman spectroscopy for tissue imaging. However, this patent only names small deuterated molecules for labeling, and is meant more for imaging and contrasting agents. Quantification by the reagents is not described.
An immunoassay displacement method for detecting the presence of or amount of a target analyte was described by White, P. C., et al. (U.S. Pat. No. 6,750,065). An antibody is bound to an analyte analog, which is labeled with a Raman active tag. The labeled analog is replaced by the analyte in an unknown sample. The released labeled analog interacts with a SERS or SERRS substrate and generates Raman signal. This method was envisioned as a drug detection and quantification method. However, the method is limited to a very specific format of immunoassay.
Another patent (EP Patent No. 0587008 to Tarcha, P. J.) described a composition, a kit, and a device for the determination of the presence or amount of an analyte. The composition comprises a specific binding member, a Raman-active label, and a substrate having a surface capable of inducing a surface-enhanced Raman light scattering. The test mixture was illuminated with a radiation sufficient to cause the Raman-active label in the test mixture to emit a detectable Raman spectrum. The differences in the detected surface-enhanced Raman scattering spectra depend upon the amount of the analyte present in the test mixture. Thus, by monitoring these differences, the presence or amount of the analyte are determined. This patent does not discuss multiplexing analysis.
Another approach was disclosed in U.S. Pat. No. 7,192,778 (Natan, M. J). Metal nanoparticles were associated with Raman-active dyes and surrounded by an encapsulant. The metal nanoparticles were used as sensitive optical tags detectable by SERRS.
Isotopic variants of Rhodamine 6G have been used for relative quantification (PCT Publication No. WO 2006/037036). It is desirable to extend the approach to more isotopic variants of Rhodamine dyes and other classes of dyes.